Use of iron oxide particles in the form of injectable aqueous suspensions for diagnostic purposes has attracted much interest in the past. Ferromagnetic species or superparamagnetic magnetite microcrystals have been used as contrast agents for the nuclear magnetic resonance imaging (MRI) of the liver and spleen. Their use as contrast agents in these organs is based on the observation that soon after injection the particles are recognized by the RES and rapidly captured. The particles are then removed from the bloodstream, stored in the liver and spleen and subsequently eliminated. Much effort has been devoted toward improvement of the known formulations with the aim to increasing the uptake of the superparamagnetic particles in the targeted organs e.g. liver, spleen or bone marrow prior to their elimination from the body thus rendering their use more practical.
With the development of NMR analysis, it has been recognized that a considerable improvement of the technique and an important advance in the art would be achieved by providing a contrast agent whose properties would enable use of NMR analysis on the entire body and not only on certain of its parts. For this purpose, however, it would be necessary to produce a medium whose magnetic properties are at least as good as those of the iron oxide particles known to date but having a residence time in the blood stream which outlasts any other particle known in the art. Hence, in this regard the problem to be solved was to find a particle which for a given period of time would not be recognized by the RES. Yet, it has been reported that improved iron oxide particles may be obtained through the use of various coating materials which when applied thereto, either modify residence times or assist the delivery of the particles to other specific sites in the animal or human body.
EP-A-0 272 091 (VESTAR) discloses coating solid particles of an active ingredient, i.e. magnetite (and other diagnostic agents or drugs) said ingredient constituting the core of the particles, with a first layer of a monomolecular amphiphile which can associate with the ingredient of the core; then, the system comprises a second outer layer, which may include a bimolecular layer of phospholipids (i.e. a liposome membrane analog) which encapsulates the amphiphile. In the examples magnetite particles coated with palmitic acid as surfactant were encapsulated in liposomes made from a mixture of cholesterol and distearoylphosphatidylcholine. One object of the arrangement is to stabilize the active ingredient in the circulation against removal.
EP-A-0 275 285 (ADVANCED MAGNETICS) discloses coated and uncoated magnetite particles for use as a contrast agent for NMR imaging. When coated, the particles are surrounded by a polymer to which biologically active molecules may be attached. In the case of coated particles, the biological molecules can be chosen to target specific organs or tissues. Polymeric coatings disclosed may be made from proteins such as albumin, polysaccharides such as dextran, polypeptides such as polyglutamates or polylysines or organosilanes such as N-2-aminoethyl-3-aminopropyltrimethoxy-silane. Biological molecules that may be covalently attached to the coating are antibodies, carbohydrates or hormones which may enhance specificity and biodistribution of the particles to specific sites in the organism.
EP-A-0 354 855 (TERUMO) discloses liposomes as drug-carrier vesicles containing polyethylene glycol bound phospholipid in the lipid layer of the vesicle. The hydrophobic moiety of the phospholipid is sunk in the membrane-constituting lipids or is bound thereto, while the hydrophilic moiety of the polyethylene glycol protrudes therefrom and extends into the surrounding medium. The liposomic vesicles are said to be useful for preparation of artificial erythrocytes by encapsulation of hemoglobin in the vesicles.
U.S. Pat. No. 4,904,479 (ILLUM) discloses coating polystyrene particles with amphiphilic block copolymers having simultaneously hydrophilic and hydrophobic segments (e.g. Poloxamer.RTM. and Poloxamine.RTM.). The coating is intended to minimize opsonization after injection and enable directing the particles to the bone marrow rather than to the liver or spleen. Poloxamer.RTM. and Poloxamine.RTM. are amphiphilic block copolymers comprising consecutive hydrophobic polyoxypropylene segments and hydrophilic polyoxyethylene segments; it is believed that for protection against uptake by the liver, the hydrophilic segments stick out from the surface of the particle outer coating, thus sterically preventing the deposition thereto of opsonin and making the particles less recognizable by the macrophages.
Although the methods of the prior art have merit, they deal with only a very specific problem of particles which can be selectively targeted to different but specific sites (e.g. liver, spleen, lungs, lymph nodes, bone marrow, etc.) whereas the present invention is set out to solve a problem of production of particles which will not be recognized by the RES, which will remain in the blood for prolonged periods of time and which would be useful for production of long lasting blood pool agents.
The particles of the prior art require expensive manufacturing techniques and produce particles which, upon injection, are recognized by the RES and easily removed from the blood. Such particles and the contrast agents produced therefrom cannot be used in applications for which a relatively long biological half-life is required.
Contrast agents with prolonged presence in the blood i.e. good resistance to uptake by RES and a relatively low diffusivity into the tissue or extravascular spots are recognized in the art as particularly useful "blood pool" agents. Long biological half-lifes are sometimes desirable for the blood pool agents if one wants to produce meaningful analytical results eliminating repeated injections and heavy use of contrast media. For obtaining such long lived blood pool agents, it would be necessary to produce "stealth" particles which, for a period of time, would not be recognized by the RES and which would still provide sufficient magnetic relaxation response. Existence of a real "stealth" iron oxide particle would enable NMR analysis of the body as a whole and not only analysis of localized parts or specific organs, as done with contrast agents known so far. The stealth particles would thus make possible measurements of blood volumes and the blood perfusion of various organs, including brain, using non-invasive techniques. For instance, monitoring variations in blood oxygenation of the brain cortex during activation tasks would become possible.
Also, particles which could remain in the blood stream for long periods would provide very valuable information on the cell status and distribution of nutrients in various organs under different physical conditions. The contrast agents made with such particles would enable direct insight into blood microcirculation and metabolic cycles of cells in the body and would therefore, open new avenues to better understanding of processes in living organisms leading to better detection of anomalies such as growth of tumors. However, the prior art has not yet succeded in providing a long lasting magnetite particles and, therefrom, blood pool contrast agents which would enable these analytical techniques to perform such measurements and to produce the desired data.